Retroviral vectors were among the earliest viral vectors developed for mammalian gene transfer. A number of retroviral species have been developed into vectors, notably alpharetroviruses (1), gammaretroviruses (2), the lentivirus human immunodeficiency virus type 1 (HIV-1) (3), nonhuman lentiviruses (4), and spumaviruses (5).
The most important structural change in moving from retrovirus to retroviral vector is the separation of viral non-coding sequences required in cis on the nucleic acid undergoing gene transfer from the viral protein coding sequences required in trans in the producer cell for the production of virions. This separation renders the vector capable of only one round of infection as no viral proteins are produced in target cells.
A standard third generation HIV-1-based lentiviral vector such as RRL or CCL (3) requires co-transfection of four plasmids into producer cells during virion production (FIG. 1): a transfer vector containing the essential cis elements and the transgene expression cassette, a packaging plasmid expressing the HIV-1 polyproteins Gag and Gag-Pol, a plasmid for the expression of the HIV-1 Rev protein, and a plasmid for expression of the viral envelope protein. Lentiviral vectors are able to incorporate envelope proteins from other enveloped viruses if they are co-expressed in producer cells, a phenomenon known as pseudotyping. The most commonly used envelope protein is the vesicular stomatitis virus glycoprotein (VSVG) which confers stability and broad tropism upon lentiviral vector virions (6).
The essential cis elements contained within the transfer vector include the HIV-1 long terminal repeats (LTRs), the RNA packaging signal (Ψ), and preferably the Rev Response Element (RRE). The LTRs contain sequences required for transcription, reverse transcription, and integration of the vector genome. In self-inactivating (SIN) transfer vectors almost all of the viral 3′ U3 region has been removed in order to eliminate its promoter and enhancer activities (3). The mechanism of reverse transcription is such that both proviral U3s originate from the 3′ LTR of the RNA genome, so proviruses resulting from infection with this vector lack LTR-driven transcription and cannot transcribe their full genome efficiently in target cells. The RNA packaging signal is thought to extend into the beginning of the gag coding sequence, but a point mutation has been introduced downstream of the gag start codon to prevent translation of the majority of this sequence. The Rev protein interacts with the RRE in producer cells to stabilise transcripts, promote RNA export from the nucleus, and enhance RNA packaging into virions (7; 8). Non-essential but commonly used cis elements include an HIV-1-derived central polypurine tract (cPPT) which enhances transduction of non-dividing cells (9) and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) which enhances viral titre and transgene expression through improving the efficiency of polyadenylation (10).
In a standard third generation lentiviral vector these cis elements are reverse transcribed along with the transgene expression cassette in target cells, resulting in a provirus incorporating two SIN LTRs containing 236 bp of HIV-1 DNA in each, a primer binding site (PBS) to Gag region containing 490 bp of HIV-1 DNA, an RRE containing 858 bp of HIV-1 DNA, and a Nef to polypurine tract (PPT) region containing 69 bp of HIV-1 DNA, making a total of 1889 bp of HIV-1 DNA delivered to target cells. These viral cis elements are covalently attached to the transgene expression cassette and persist in target cells after transduction. If an integrating lentiviral vector is used, they are irreversibly integrated into target cell chromosomes.
Consequences of Transfer Vector cis Element Persistence in Target Cells
The long term persistence of HIV-1-derived cis elements in target cells creates a number of experimentally observed and theoretically possible problems for the practical application of lentiviral vectors in studying the biological effects of transferred genes on cell culture or animal models, the generation of transgenic animal strains and stable cell lines, and the transfer of therapeutic genes to treat human disease.
Firstly, the transfer vector cis elements contain active splice acceptor sites which are able to splice with host cell genes to create aberrant fusion transcripts (11-13). An event of this type was observed in a gene therapy clinical trial in which splicing between a copy of the patient's growth-promoting HMGA2 gene and an integrated lentiviral provirus caused dysregulation of HMGA2 transcription and a large clonal expansion of a transduced cell (14).
Secondly, the persistence of cis elements required for RNA packaging enables remobilisation of self-inactivating lentiviral vector genomes in cells expressing viral proteins (15). If used in patients infected with HIV this could result in remobilised lentiviral vector proviruses and recombination with wildtype HIV-1 genomes.
Thirdly, lentiviral cis elements contain untranscribed CpG-rich DNA which is subject to DNA methylation and may contribute to reduction in expression of the delivered transgene through silencing in host cells (16).
Fourthly, large untranscribed regions within episomal DNA vectors have been associated with transgene silencing in vivo (17). Reducing the size of the untranscribed region within an episomal integration-deficient lentiviral vector (IDLV) may therefore improve long term expression in applications of these vectors.
Fifthly, reducing the size of the reverse transcript may increase the transgene carrying capacity of lentiviral vectors. There are potentially multiple stages of the lentiviral life cycle which are limiting for vector genome size, such as RNA packaging (18), reverse transcription in cell types with low intracellular dNTP concentration (19), and integration into chromosomes.
Minimisation of cis Elements within Target Cell Proviruses
Several approaches have been taken towards the goal of minimising the viral cis elements which persist within target cell proviruses.
Firstly, a number of authors have investigated the effect of simple deletions and point mutations to remove or inactivate the remaining cis elements. Cui et al reported point mutations to the HIV-1 major splice donor (MSD) positioned upstream of Gag as well as further incremental deletions of the Gag and RRE elements resulting in a transfer vector containing 550 bp of HIV-1 cis DNA (or 786 bp if both LTRs in the provirus are accounted for) (20). In 293T producer cells these changes resulted in a large decline in expression of unspliced transfer vector RNA, but in TE671 cells this effect was less pronounced due to cell type-specific splicing patterns and the resulting decline in titre was only 2-fold. This system has not been widely adopted within the field, perhaps due to a combination of the reduction in titre and the unusual producer cell line. Kotsopoulou et al reported an attempt to generate a Rev-independent lentiviral vector by codon optimisation of the packaging plasmid and deletion of the RRE from the transfer vector, resulting in a 5-fold reduction in titre (21). It has since been reported that the Rev/RRE system is required for efficient packaging of transfer vector RNA into virions (8). Koldej et al reported minimisation of the amount of Nef coding sequence upstream of the PPT (22). The entire sequence appears to be dispensable up to the run of 5 thymidine bases immediately upstream of the PPT.
A second approach towards the minimisation of cis elements within target cell proviruses is to design vectors in which these elements are present in the viral RNA genome but subsequently deleted during reverse transcription or integration.
Delviks et al reported a gammaretroviral vector in which the RNA packaging signal was flanked by a 701 bp repeat of the herpes simplex virus thymidine kinase gene (HSV-TK) (23). During reverse transcription in target cells, template switching by the reverse transcriptase from one repeat to the other resulted in the deletion of the RNA packaging signal. Since template switching is not an obligatory activity for the reverse transcriptase, the efficiency of this deletion was reported to be 91% of clones. This strategy to delete cis elements has the disadvantage of requiring a new cis element to be introduced (in this case, HSV-TK) which is not itself deleted. Patents and patent applications derived from this vector include U.S. Pat. No. 5,741,486, U.S. Pat. No. 5,714,353 and WO 95/032298. A similar strategy was used by Srinivasakumar in a lentiviral vector in which the RRE was flanked by repeat copies of the hygromycin phosphotransferase gene, resulting in deletion of the RRE in 84% of clones (24).
Torne-Celer et al used the ability of retroviral integrases to cleave internal att sites to generate alpharetroviral vectors in which the 5′ LTR and the RNA packaging signal are cleaved off the pre-integration complex by the viral integrase enzyme during the 3′ end processing stage of integration (25). While 61% of clones in this report carried the expected deletion, internal att site processing appears to produce heterogeneous proviral products and results in titres that are 102 to 103-fold lower than would be expected with a more conventional alpharetroviral vector (26).
A third approach towards the minimisation of lentiviral cis elements is to remove them from target cell proviruses following transduction. Luche et al reported a lentiviral vector in which the RNA packaging signal was flanked with loxP sites so that it could be excised when Cre recombinase was provided in trans in target cells (27). In transduced 293T cells the excision was successful in 12-20% of transduced cells. Fang et al reported a self-minimising lentiviral vector incorporating a Cre recombinase expression cassette which excised the RNA packaging signal, RRE and itself following target cell transduction (28). Successful excision as measured by loss of Cre expression took place in 92% of target cells. Both of these strategies rely on the successful expression and function of Cre recombinase in target cells, and it is highly unlikely that such a strategy would receive regulatory approval for use in patients in the near future.